The chloroplast genome of Chrozophora sabulosa Kar. & Kir. and its exploration in the evolutionary position uncertainty of genus Chrozophora

Chrozophora sabulosa Kar. & Kir. is a biennial herbaceous plant that belongs to the Euphorbiaceae family and has medicinal properties. This research aimed to identify the genetic characteristics and phylogenetic position of the Chrozophora genus within the Euphorbiaceae family. The evolutionary position of the Chrozophora genus was previously unknown due to insufficient research. Therefore, to determine the evolutionary link between C. sabulosa and other related species, we conducted a study using the NGS Illumina platform to sequence the C. sabulosa chloroplast (cp.) genome. The study results showed that the genome was 156,488 bp in length. It had a quadripartite structure consisting of two inverted repeats (IRb and IRa) of 24,649-bp, separated by an 87,696-bp LSC region and a 19,494-bp SSC region. The CP genome contained 113 unique genes, including four rRNA genes, 30 tRNA genes, and 79 CDS genes. In the second copy of the inverted repeat, there were 18 duplicated genes. The C. sabulosa lacks the petD, petB, rpl2, and rps16 intron. The analysis of simple sequence repeats (SSRs) revealed 93 SSR loci of 22 types and 78 oligonucleotide repeats of four kinds. The phylogenetic investigation showed that the Chrozophora genus evolved paraphyletically from other members of the Euphorbiaceae family. To support the phylogenetic findings, we selected species from the Euphorbiaceae and Phyllanthaceae families to compare with C. sabulosa for Ks and Ka substitution rates, InDels investigation, IR contraction and expansion, and SNPs analysis. The results of these comparative studies align with the phylogenetic findings. We identified six highly polymorphic regions shared by both families, which could be used as molecular identifiers for the Chrozophora genus (rpl33-rps18, rps18-rpl20, rps15-ycf1, ndhG-ndhI, psaI-ycf4, petA-psbJ). The cp. genome sequence of C. sabulosa reveals the evolution of plastid sequences in Chrozophora species. This is the first time the cp. genome of a Chrozophora genus has been sequenced, serving as a foundation for future sequencing of other species within the Chrozophoreae tribe and facilitating in-depth taxonomic research. The results of this research will also aid in identifying new Chrozophora species. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10366-3.

The family Euphorbiaceae is challenging to understand due to its wide distribution range, many species, and poorly defined genera.However, it has been confirmed that Euphorbiaceae is monophyletic based on molecular and embryological features [8][9][10].Webster [6] has classified the Euphorbiaceae family into five subfamilies based on the number of ovules per ovary locule: Phyllanthoideae, Oldfieldioideae, Acalyphoideae, Crotonoideae, and Euphorbioideae.In the Angiosperm Phylogeny Group (APG II 2003) [11] classification, the family was divided into four groups: Euphorbiaceae s.s., Phyllanthaceae, Picrodendraceae, and Putranjivaceae, all in the clade Malpighiales.The subfamilies with uniovulate ovary locules (Euphorbioideae, Acalyphoideae, and Crotonoideae) are considered Euphorbiaceae s.s [11].The family was further divided into several subfamilies and tribes based on molecular data [12], and some genera were moved to independent families [5].APG III (2009) [13] divides it into four subfamilies: Acalyphoideae, Cheilosoideae, Crotonoideae, and Euphorbioideae.Despite the extensive research carried out by botanists, who have conducted studies in taxonomy, anatomy, phytochemistry, economic botany, and molecular systematic, the knowledge of this family still has significant gaps, even regarding morphology [5].Detailed molecular, morphological, and anatomical studies involving many genera are required to propose a safer classification for this family.However, some genera within the family, such as Chrozophora, still have confusing taxonomic positions.To understand the evolutionary relationship between C. sabulosa and related plants, it is necessary to sequence the chloroplast genomes from the Chrozophora genus and the Chrozophoreae tribe.
Previous studies have used both molecular and morphological data to perform phylogenetic reconstructions.However, it has been suggested that molecular approaches are more reliable in phylogenetics [8][9][10].Among molecular approaches, cp.genome sequences have gained significant interest in plant phylogenetics, phylogeography, and molecular evolution investigations in recent years [14].The cp. genome has a significantly conserved gene content and genome order [15].Moreover, it is smaller, has fewer nucleotide alterations, and has fewer genome sequence reorganizations than the nuclear and mitochondrial genomes.These characteristics make it an excellent tool for understanding genome evolution in complicated angiosperm families [14][15][16][17][18].As a result, cp.genomes provide valuable data that can be easily combined with source molecular data to validate complicated evolutionary connections and perform comprehensive phylogenetic analyses [17].More than 400 species of Euphorbiaceae have had their cp.genomes sequenced using a high-throughput sequencing technique [10].However, data on the chloroplast genomes of Chrozophora was unavailable, which limits further information regarding its phylogenetic position in the family.Additionally, no members of the Chrozophoreae tribe have had their cp.genomes sequenced yet, which casts doubt on the tribe's exact phylogenetic position.The present study aimed to investigate the cp.genome of C. sabulosa to clarify the evolutionary position of the Chrozophora genus in the Euphorbiaceae family.By contributing significant molecular and phylogenetic data on the Chrozophora genus, this study may aid in species identification and determination of its evolutionary position.The C. sabulosa cp.genome has been marked as the first member of the Chrozophora genus and the Chrozophoreae tribe to be sequenced.The results of this research may provide a foundation for phylogenetic investigations of the Chrozophoreae tribe.was 36.5%, with the highest GC content (43.3%) found in the IRs, followed by 34.1% in the LSC region and 30% in the SSC region.The CP genome contained 113 distinct genes, including 79 CDS genes, four rRNA genes, and 30 tRNA genes, as shown in Table 1.The LSC segment had 84 genes, while the SSC segment had 13.Table S1 listed the 15 genes (out of 113) with introns, including three with two introns (clpP, ycf3, rps12), five tRNA, and five CDS genes with one intron.The rps12 gene was repeated twice, leading to a trans-splitting event.The psbL gene in C. sabulosa began with a TCG codon, resulting in Threonine as the first amino acid.The cp. genome lacked the Fig. 1 The cp. genome map of C. sabulosa.Genes outwards the map is demonstrated clockwise, while inside genes are transcribed anticlockwise.Color coding is used to differentiate between functional groups of genes.The intensity of the inner circle color indicates the amount of GC (Dark grey) and AT (Light grey).LSC denotes the large single copy, SSC denotes the small single copy, and IRb and IRa denote inverted repeats petB, petD, rpl16, and rpl2 introns.Table 2 provides a detailed description of the genes in C. sabulosa based on their function.

RSCU, and amino acid frequencies in C. sabulosa
All genomes showcase codon usage bias, which affects translational dynamics, consistency, accuracy, and protein folding [19].The Relative synonymous codon usage (RSCU) ratio is the average usage frequency of a codon divided by its predicted unbiased usage frequency.Recent studies have highlighted the significance of codon usage in the evolution of the cp.genome [20,21].In the coding sequences in C. sabulosa, there are 52,162 codons within 79,461 bp.The most abundant amino acid in the cp.genome of C. sabulosa is leucine (11%), followed by isoleucine (9%), and the least abundant is cysteine (1%) (for further details, see Table S2 and Fig. 2).We have identified 31 variant codons with RSCU values greater than one, indicating that C. sabulosa uses them specifically to encode certain amino acids.The AGA codon, which codes for arginine, has the highest usage bias (2.05), while the CGC codon, which also codes for arginine, has the lowest (0.44).There was no bias at codons AUG (Methionine), CCC (Proline), and UGG (Tryptophan) in C. sabulosa cp.genomes with 1.00 RSCU (Table S3).

Editing sites of RNA
PREP-cp detected 50 RNA editing sites in 21 genes from C. sabulosa.The ndhB gene has the highest number of  RNA editing sites (9 sites), followed by the ndhD gene (7 sites) (refer to Fig. 3).Modifying a nucleotide in the first position of a codon resulted in 13 editing sites (26%), while changes in the second position resulted in 37 editing sites (74%).Most RNA editing sites (42%) were found in Serine codons.In C. sabulosa, 90% of the Serine was converted to Leucine, while the remaining 10% was converted to Phenylalanine.The codon-encoded Proline had the second-highest conversion rate (18%), while the codon-encoded Threonine had the third-highest conversion rate (14%).Proline, Serine, Threonine, and Alanine showed multiple types of nucleotide conversion, whereas Leucine, Arginine, and Histidine showed only one.Hydrophobic amino acid conversions [including Proline (9), Alanine (2), and Leucine ( 6)] occurred at 34% of all RNA editing sites.In contrast, soluble amino acids resulted in 33 conversions (66%), including Histidine (3), Threonine (7), Serine (21), and Arginine (2).Three nonpolar to polar conversions, 14 non-polar to non-polar conversions, 30 polar to non-polar conversions, and three polar-to-polar amino acid conversions have also been found.For further information, please refer to Table S4, which details all the RNA editing sites.

Oligonucleotide repeats analysis of C. sabulosa
We used the REPuter tool to identify 79 oligonucleotide repetitions, which had the following values: P = 27, F = 27, C = 7, and R = 18, as illustrated in Fig. 5.These repeats had a size ranging from 20 to 51 bp (as shown in Fig. 5b).We found that the LSC contained 57 oligonucleotide repeats, the SSC had 11, and the IRs had four.Furthermore, we discovered that IR and LSC shared three repeat layouts, with two shared by LSC/SSC and one shared by SSC/ IR (as depicted in Fig. 5c).The number of oligonucleotide repetitions in intergenic spacer areas was highest (55), followed by CDS ( 9), transfer RNA (3), and intronic region (2).We also detected mutual repeats in the IGS/ CDS (2), IGS/Intron (4), and IGS/trn (3) regions (as shown in Fig. 5d).Palindromic repeats were more frequent than other repetitions (as seen in Fig. 5a).The locations, positions, and areas of oligonucleotide repeat sequences are provided in Table S8.

Phylogenetic analyses
We created a maximum likelihood tree using CDS data from Euphorbiaceae and Phyllanthaceae cp.genomes (Fig. 6, Table S12) to investigate the evolutionary relationship of Chrozophora.We constructed the tree by using C. sabulosa, 18 other genera from Euphorbiaceae, and 10 genera from Phyllanthaceae.The alignment of 31 species using MAFFT produced an

The expansion and contraction of IRs
A study was conducted on the margins of four key areas (LSC, IRB, SSC, IRA) and their surrounding genes in C. sabulosa and selected cp.genomes (Fig. 7).The ycf1 gene on the JSA (SSC/IRA) junction was found to be functional in all species.However, in R. communis, M. esculenta, J. curcas, E. helioscopia, (A) bunius, and G. chodoense, a pseudo copy of ycf1 was detected at the JSB (IRB/SSC) border, while it was absent in C. sabulosa, (B) fruticosa, and P. urinaria.The size and position of the ndhF gene varied at the JSB border.The rps19 and rpl2 genes were entirely in the IRs in (C) sabulosa, E. helioscopia, A. bunius, and P. urinaria, but they were found in varied locations in the other five species.The trnH gene was detected at the JLA (IRA/LSC) boundary in all species except A. bunius, which had two copies of this gene found in IR regions.The study revealed that identical genes varied in locations and sizes at every junction of the cp.genomes, indicating a variety of gene content.
A thorough analysis of IR contraction and expansion is shown in Fig. 7 (IRSCOPE analysis).These findings suggest that these nine cp.genomes were slightly different due to differences in size and gene placement in these species.

The Ka, Ks substitutions, and Ka/Ks rate
Pair-wise alignments of C. sabulosa genes were performed with eight selected cp.genomes to determine the Ka/Ks ratio (Fig. 8).All comparisons of selected genes with all species had a Ka/Ks ratio that was typically less than one.Genes for which Ka/Ks ratios were unavailable (N/A) were set to zero (See Table S10).After excluding the genes with a Ka or Ks value of zero, the average Ka/ Ks ratio was 0.20, demonstrating that the genes in the cp.genome of C. sabulosa were subjected to significant purifying selection forces.The average Ka/Ks ratio for Euphorbiaceae species was 0.212, while it was 0.184 for the Phyllanthaceae species.The psbI and petN have zero Ka/Ks ratio in all species, making them the most stable genes among both families.Most of the genes exhibited a Ka/Ks ratio of below one in all comparisons, and their proportions were consistent, except for petD, ndhK, cemA, rpl23, and rpl20 genes, which had challenging ratios.For instance, in R. communis, E. helioscopia, and G. chodoense, the Ka/Ks rate of petD was above one, but in the other five comparisons, it was less than one.Compared to R. communis, the Ka/Ks ratio of petD was 9.1, whereas it was only 0.03 when compared to J. curcas.
Similarly, the Ka/Ks value of ndhK was 1.12 compared to R. communis.The Ka/Ks values of the five challenging genes are displayed in Table 4.The comprehensive Ka and Ks values and their ratios are available in Supplementary Table 9.

Investigating SNPs and InDel mutations analysis in C. sabulosa
We conducted pair-wise alignments between C. sabulosa and selected species of Euphorbiaceae and Phyllanthaceae families.As a result, we discovered single nucleotide polymorphisms (SNPs) and InDels mutation events in the IR, SSC, and LSC regions of C. sabulosa.The highest number of SNPs (18,625) was found when comparing C. sabulosa to E. helioscopia, while the lowest number was found when compared to M. esculenta (17,028 SNPs).All species in Table 5 had a transition-to-transversion ratio greater than one due to more transitions than transversions.The LSC and SSC regions showed a higher rate of substitutions than the IR regions.In terms of inDels, the inverted repeat regions had the fewest, while the SSC and LSC regions had the most.Pair-wise alignment of J. curcas resulted in the most significant number of inDels (24,369), followed by P. urinaria with 23,217 inDels, and E. helioscopia with the least (21,134).Table 6 provides an in-depth description of inDels and its relevant parameters.The similar values of InDels and SNPs observed in all the Euphorbiaceae and Phyllanthaceae species indicate their closer relationship.The nucleotide diversity and highly polymorphic loci of Euphorbiaceae and Phyllanthaceae species, with particular reference to C. sabulosa We conducted an independent analysis by comparing C. sabulosa with representatives of both Euphorbiaceae and Phyllanthaceae families.We aimed to examine the nucleotide diversity and highly polymorphic loci for C. sabulosa and other chosen species of these two families.We found that the average nucleotide diversity in Euphorbiaceae species was 0.0958, whereas in Phyllanthaceae species, it was 0.0981.The nucleotide diversity values varied across species and regions.In Euphorbiaceae species (Table S10), it ranged from 0.0076(rps7) to 0.2549(rpl33-rps18), whereas in Phyllanthaceae species (Table S11), it ranged from 0.0068(rps7) to 0.2467(rps15-ycf1).We observed that the average nucleotide diversity in coding areas was the lowest (Euphorbiaceae species 0.0624, Phyllanthaceae species 0.0652), followed by IGS regions (Euphorbiaceae species 0.1934, Phyllanthaceae species 0.1953), and Intronic regions (Euphorbiaceae species 0.6178, Phyllanthaceae species 0.6193).Additionally, we identified six highly polymorphic sites shared by both families (Table 7), which can be used as mutational markers to identify and classify Chrozophora species.Figure 9 shows the nucleotide diversity values for the 93 locations selected from both families.We found that C. sabulosa had identical nucleotide diversity values with both family members, indicating that it shares characteristics of both families.

Discussion
We are excited to present our novel findings on the chloroplast genome of C. sabulosa, a first-time report in the scientific community.Our research on the chloroplast genome of C. sabulosa unveiled unique and intriguing findings.The TCG codon in the psbL gene of C. sabulosa leads to Threonine as the first amino acid, a behavior similar to that observed in the cp.genomes of other plant species such as Indigofera genus [27], Spinacia oleracea (NC_002202), Nicotiana tabacum (NC_001879), Ampelopsis glandulosa (KT831767), Lycium barbarum (MH032560), and Lycium chinense (MK040922).This underscores the conserved structure of the chloroplast genome, a feature observed in various other angiosperm lineages [17,[28][29][30][31].Our findings also revealed that the DNA GC percentage is not uniform within the chloroplast genomic domains, with the GC concentration in the IR area being more significant than that of the other regions, likely due to the high GC concentration found in the four rRNAs in the inverted repeats [31].Furthermore, our comparison of the cp.genome of C. sabulosa with other species in the Euphorbiaceae and Phyllanthaceae families yielded significant findings.The cp. genomes and gene content were similar across selected species.However, the infA gene was only present in C. sabulosa and (A) bunius and was missing in other Euphorbiaceae and Phyllanthaceae cp.genomes.The pseudo copy of ycf1 was absent in C. sabulosa, (B) fruticosa, and P. urinaria.These findings highlight the unique gene content and organization in (C) sabulosa and its evolutionary implications [32][33][34][35][36][37].We also discovered that the petD, petB, rpl16, and rpl2 genes of C. sabulosa and some other species lacked introns, a phenomenon documented in various other angiosperms [38,39].The genes in which intron loss was reported earlier in other angiosperms are rpoC2, atpF, rpl2, rps12, atpF, rps16, and clpP [38][39][40][41][42].This underscores the critical role of introns in gene expression control and their potential to boost exogenous gene expression at plant genome regions to achieve desirable agronomic features [37].The lack of specific introns may cause changes in gene expression [37].
The codon usage bias in the cp.genome of plants is an essential evolutionary characteristic that affects the translation of mRNA, gene identification, and molecular biological investigations [32].Some genes in plastoms have shown a bias towards specific codons, likely due to external pressure [43].C. sabulosa's cp.genome uses leucine most commonly and cysteine uncommonly.Similar findings have been reported in other cp.genomes, such as Eruca sativa [43], Farsetia hamiltonii [17], and Nasturtium officinale [44].AGA codon in C. sabulosa's cp.genome had the highest usage bias for Arginine.These findings suggest that codon usage significantly impacts the reshaping and translation of the cp.genome [17,[19][20][21][43][44][45][46][47].Our results also support earlier studies on the adaptational evolution of the large A/T concentration in chloroplast genomes, which have also shown a preference for specific codons [17,19,22,23].
RNA editing is a modification after transcription, significantly impacting the sequencing and performance of related proteins and genetic material [45].Analyzing the RNA editing sites in C. sabulosa could provide evolutionary insights into how RNA editing systems evolved during the evolution of plant life on Earth and which editing sites may have been maintained to carry out essential functions.Most RNA editing sites were found in the ndhB gene, which encodes for NADH dehydrogenase subunits.This demonstrated that a single gene could translate a wide range of protein products using RNA editing [41].Changes at the second position of the nucleotide were more prevalent than changes at   other positions among the RNA editing sites examined.RNA editing, particularly at the second codon position, can alter the encoding amino acid and the primary, secondary, and tertiary protein organization, which may be essential for their function [41,45].Most of the RNA editing sites were discovered in Serine codons, with the most significant transformation of Serine into Leucine, possibly increasing the hydrophobicity of the associated peptide.Our findings also supported that RNA editing sites can restore amino acid conservation, improve hydrophobicity, and impact protein architecture [41].These findings were consistent with the fundamental properties of chloroplast gene RNA editing in higher plants [17,41,45].
Our study on the C. sabulosa cp.genome revealed that mono-nucleotide repeats were the most common and pentanucleotide was the most extended SSR type.Our results were consistent with similar studies on angiosperm species, indicating that polyadenine and polythymine repeats are more abundant in cp.genomes [17,26,29,44,51,52].We did not observe any hexanucleotide SSRs in the C. sabulosa cp.genome, which is a shared trait with Brassica napa, Nasturtium officinale, Raphanus sativus, and Fritillaria cp.genomes [44,48,49].Singlecopy regions had a higher percentage of oligonucleotide repeats than inverted repeats, confirming the reverse nature of inverted repeats [17].The IGS had more repeats than other cp.genome regions, indicating higher susceptibility to mutations and recombination [17,29].Palindromic repeats were more frequent than other types of repetitions, suggesting the existence of various identical or comparable sequences, either continuous or separated by a spacer region [17,28,29].Our findings were consistent with several studies conducted on angiosperms [17,28,29,37,[53][54][55].
High-dimensional sequencing methods have made it easier to access CP genomes with vast amounts of genetic material [17,43].For phylogenetic research, cp.genome sequences are an excellent resource [17,43,53,56,57].The Euphorbiaceae family is one of the most diverse angiosperm families [4,5,58].However, there is conflict in sub-famil classification within the Euphorbiaceae family.Previously, based on pollen morphology, the Euphorbiaceae was classified into five sub-families, including Phyllanthoideae, Oldfieldioideae, Acalyphoideae, Crotonoideae, and Euphorbioideae [6].Later, the Angiosperm Phylogeny Group [13,59] separated the Phyllanthaceae from the Euphorbiaceae, giving it a separate family status.Recently, Euphorbiaceae has been divided into four subfamilies based on molecular data [59,60].However, this classification is also unclear due to a lack of available data on its species.Chloroplast genomes have been used to determine the phylogenetic relationships in the Euphorbiaceae family [18,[23][24][25][26].The systematic position of Chrozophora was unclear until this study.The findings suggest that Chrozophora is closely related to the Phyllanthaceae family, which supports the historical record of the Euphorbiaceae family being the ancestor of the Phyllanthaceae family [6].The Chrozophora genus is considered to be distinct from the other Euphorbiaceae family genera, indicating its paraphyletic origin.Our results also confirmed that the Euphorbiaceae family's immense diversity, morphological divergences, variable ecological range distribution, and the scarcity of literature on numerous species make phylogenetic interpretations challenging [60].Additionally, there is a critical need to sequence more chloroplast genomes of the Chrozophoreae tribe to clarify its position among these two families.
The cp. genome is known to be stable across different plant lineages.However, the expansion and contraction of IRs can alter the size of the cp.genome and its segments [17,28,53,61,62].The expansion and contraction of IRs affect genes, substitutions, and genome length, ultimately determining a species' phylogenetic position [17,34,63].Previous studies have found that changes in the boundaries of the cp.genome are caused by differences in the number and location of genes at the interface of inverted repeats [17,34].We studied the IR regions of C. sabulosa, four Euphorbiaceae, and four Phyllanthaceae species.Our findings revealed both similarities and genetic variations in these plastomes.The conservation of the IR region was higher in all plastomes, while most substitutions occurred in the LSC and SSC regions.These results are consistent with similar studies on other plastid genomes [17,28,[62][63][64].Our research also showed that gene migration between single copies and inverted repeat regions causes mutation rate variations, making chloroplast genomes either conserved [17,28,31,34] or widely polymorphic in gene content and structure [64][65][66][67].
The Ka/Ks ratio confirms the selective forces that have acted upon the genes during evolution.These forces can be impartial, pure, or positive depending on the Ka/Ks proportion [17,68,69].We compared the cp.genome of C. sabulosa to eight selected species to validate our phylogenetic findings.Our analysis showed that most C. sabulosa genes underwent purifying selection to maintain their preserved function.Purifying selection on most chloroplast genes in the Euphorbiaceae and Phyllanthaceae family species contributed to corroborating phylogenetic results.However, a few genes showed abnormal behavior regarding Ka/Ks values, indicating petD, ndhK, cemA, rpl23, and rpl20 in both family representatives.Our research findings are in collaboration with the previously published similar results [17,43,46,70,71].InDels and SNPs were more frequent in the LSC section than in the inverted repeats region.The IRs had the lowest number of mutations, indicating their conserved nature over the single-copy regions [17,28,[62][63][64].The transition-to-transversion ratio was more significant than one in all selected species, indicating a higher transition rate than the transversion rate.This suggests that the species are distant and have more SNPs [17,22].The high Ts/Tv ratio may be due to the GC-rich composition of the chloroplast genome [61]; similarly, the nuclear genomes have already been reported to have a Ts/Tv ratio due to their GC-rich makeup [72].The high number of InDels and SNPs (significant mutations) indicated Chrozophora's unique phylogenetic position and paraphyletic evolution.
We analyzed nucleotide diversity in C. sabulosa and selected species from the Euphorbiaceae and Phyllanthaceae families.Results showed that IGS regions have higher rates of genetic recombination and polymorphisms than protein-coding regions.These findings confirmed that IGS regions are more susceptible to genetic recombination and polymorphisms than protein-coding regions.These outcomes also supported the conserved status of the protein-coding genes reported earlier in other plastid genomes [17,28,31,34].Nucleotide diversity is low in Euphobiaceae and Phyllanthaceae species, ranging from 0.007 to 0.24.This suggests that the plastome architecture is conserved in both families, consistent with previous studies [17,[72][73][74][75][76].We identified six highly polymorphic regions shared by both families that could be used as molecular identifiers for the Chrozophora genus (rpl33-rps18, rps18-rpl20, rps15-ycf1, ndhG-ndhI, psaI-ycf4, petA-psbJ) all with π > 0.5.For further validation of the results of this study, more species of the Chrozophora genus and the Chrozophoreae tribe must be sequenced.

Conclusion
This is the first cp.genome of C. sabulosa, which is also the first member of the genus and tribe reported.It has a typical quadripartite structure and gene content that is quite similar to other chloroplast genomes.Our comparative analysis with other Euphorbiaceae and Phyllanthaceae species has highlighted the conserved structure of the chloroplast genome, the non-uniform distribution of GC percentage, and the unique gene content and organization of C. sabulosa.Our investigation into codon usage bias and RNA editing sites has provided insights into the evolutionary characteristics of the cp.genome and the potential impact on protein organization and function.The phylogenetic analysis in this study revealed Chrozophora's unique position in the Euphorbiaceae family, supporting the idea that this genus is paraphyletic.This chloroplast genome from C. sabulosa will be useful for the molecular characterization of related Chrozophoreae tribe species in the future.The phylogenetic data presented by this study will also aid in determining the genus' location in Euphorbiaceae family.The highly polymorphic loci identified in this study could be used as markers for future Chrozophora species identification.Furthermore, it is essential to sequence its sister Chrozophora species to fully comprehend its phylogenetic position and evolutionary dynamics.Overall, our study provides valuable information on the chloroplast genome of C. sabulosa and its evolutionary implications for the scientific community.

Collection of plant material and its sequencing
Nida Javaid and Shagufta Jabeen conducted the formal plant material identification for this study.Fresh leaves of Chrozophora sabulosa plants were collected from the Lesser Cholistan desert in Pakistan (28.7719699, 71.3346211) and the verification process was carried out at the Cholistan Institute of Desert Studies (CIDS) of the Islamia University Bahawalpur.The herbarium of plants was submitted to the CIDS for identification.A voucher number was issued for Chrozophora sabulosa Kar.& Kir.Nilkari of Euphorbiaceae which is CIDS/IUB-1601/59.For DNA extraction, the phenol-chloroform (Organic) [77] procedure was used with a few modifications.These modifications included using 1 µL 2-Mercaptoethanol and precipitating DNA with absolute ethanol following a wash with 70% ethanol.The extracted DNA was then analyzed for its quantity and quality using Nanodrop and 1% agarose gel electrophoresis.A full genome shotgun was generated by a Paired-end library of 150 bp with 350 bp insert size using Illumina Hiseq2500 at the Beijing Institute of Genomics (BIG), Beijing, China.

RNA editing site, codon usage, and amino acid frequency
We used Geneious Prime 2023.2.1 to analyze the amino acid frequency, and MEGA-X to examine Relative Synonymous Codon Usage (RSCU) in protein-coding sequences of C. sabulosa [88].Additionally, we used Predictive RNA Editors for Plants Chloroplast (PREP-cp: http://prep.unl.edu/) to find RNA editing sites in 21 protein-coding genes [89].

Phylogenetic analysis
The National Center for Biotechnology Information (NCBI) was used to select 18 species plastomes from the Euphorbiaceae family for phylogenetic analysis (Table S12).These species represent 11 tribes and three subfamilies of the Euphorbiaceae, including Acalyphoideae, Crotonoideae, and Euphorbioideae.Additionally, 10 species of the Phyllanthaceae family and two out-groups (Mangifera indica and Lannea coromandelica) from the Anacardiaceae family were chosen for analysis.In total, 31 species were included in the phylogenetic tree.The protein-coding sequences for each species were extracted and concatenated using Geneious Prime 2021.1.1.The sequences were then aligned using MAFFT in Geneious Prime 2023.2.1.The maximum likelihood tree was constructed online in Galaxy (https://usegalaxy.org)using IQ-TREE [92] and 1000 bootstrap replications with Ultrafast bootstrap settings [93].The best-fit model was selected according to the Akaike information criterion (AIC) [94].We completed the tree display using iTOL (https://itol.embl.de/#)[95].

Comparative analyses with C. Sabulosa
The cp. genomes of four species from the Euphorbiaceae family (Ricinus communis, Manihot esculenta, Jatropha curcas, and Euphorbia helioscopia) and four species from the Phyllanthaceae familys (Antidesma bunius, Breynia fruticosa, Glochidion chodoense, and Phyllanthus urinaria) were compared to that of C. sabulosa.This was done using phylogenetic analysis outcomes.The Geneious Prime 2023.2.1 was used to perform a basic comparison of the plastomes.IRscope (https://irscope.shinyapps.io/irapp/)was used to observe IR contraction and expansion in the LSC/IRB/SSC/IRA junctions among these selected species [96].Pairwise comparisons of the 78 protein-coding genes common in C. sabulosa and the eight selected species were performed to estimate synonymous (Ks) and non-synonymous (Ka) substitution rates.C. sabulosa was used as the reference member to make pairwise alignments with every gene of the selected species.Firstly, MAFFT in Geneious Prime 2023.2.1 software was used to perform 624 pairwise alignments of the identified genes among species [82,83], and then DnaSP [97] was employed to examine Ka and Ks substitutions.Geneious Prime 2023.2.1 was used to calculate the number, coordinate placements, and types of substitutions (transition and transversion).DnaSP [97] was used to find InDels mutations for each part of the pairwise aligned cp.genomes.The alignment length, inDel average length, k(i) inDel diversity, and Pi(i) inDel diversity per site were also calculated.

Nucleotide diversity to determine highly polymorphic loci
We analyzed to compare Nucleotide diversity (π) values among representatives of both families along with C. sabulosa.We extracted a total of 837 regions, which included 59 CDS genes, 27 IGS regions, and seven intronic regions that were common in all species.To create multiple alignments of 93 locations of C. sabulosa with members of both families separately, we used MAFFT [82].We only selected sequences that were longer than 200 base pairs [98].To calculate the Nucleotide diversity (π), we used DnaSP [97].We identified six highly polymorphic loci that had greater nucleotide diversity to compare among the selected species [98].

Fig. 9
Fig. 9 Nucleotide diversity (π) in 93 regions common in Euphorbiaceae and Phyllanthaceae family members.The π values of Euphorbiaceae members are displayed in sky blue, whereas the π values of Phyllanthaceae members are depicted in red

Table 1
The cp. genome of Chrozophora sabulosa is described in detail

Table 2
Chrozophora sabulosa gene functions are summarized in this table of both families and showed that the members of the Phyllanthaceae family share a molecular basis with members of the Euphorbiaceae family.Additionally, our analysis revealed Chrozophora's unique position within the Euphorbiaceae family.
80,283 bp consensus sequence with 22,018 (22.5%) identical positions and 84.8% pair-wise identity.The tree comprised 28 nodes with bootstrap values ranging from 48 to 100.The bestfit model for this tree was GTR + F + R6.Its log-likelihood was − 471155.9363,AICscore942465.8726,AICcscore942465.9954,andBICscore943196.6990.The overall length of the tree was 0.9525, with internal branch lengths of 0.2132 (22% of tree length).Our analysis indicated that the Chrozophora genus evolved from other Euphorbiaceae members in a paraphyletic manner.The Chrozophora genus was closely related to the Bischofia genus (Bischofia polycarpa) of the Phyllanthaceae family.This finding suggests that the Chrozophora genus is more closely associated with Phyllanthaceae members than other Euphorbiaceae members.The tree also confirmed the common ancestorC.sabulosa'scomparisonwithotherEuphorbiaceaeand phyllanthaceae speciesWe have selected eight plant species from two families, Euphorbiaceae and Phyllanthaceae, to compare their chloroplast genome with C. sabulosa.The four selected species of Euphorbiaceae are Ricinus communis, Manihot esculenta, Jatropha curcas, and Euphorbia helioscopia, while four Phyllanthaceae species are Antidesma bunius, Breynia fruticosa, Glochidion chodoense, and Phyllanthus urinaria.A detailed basic comparison is shown in Table3.We have compared the length of the chloroplast sequence and the quadripartite structure of each species.The length of the chloroplast sequence varied from 155,630 bp (B.fruticosa) to 163,856 bp (J.curcas), and each segment of the quadripartite structure was comparable across the analyzed plastomes.M. esculenta and E. helioscopia had the highest number of genes (132), while B. fruticosa and P. urinaria had the fewest (129).The overall GC content of these cp.genomes varied from 35.4 to 36.7%, and the gene component was comparable, except for a few missing or added genes.The infA gene was present in C. sabulosa and A. bunius but not in the other seven species.The rps16 gene was absent in J. curcas and E. helioscopia, while the petD intron was absent in C. sabulosa, R. communis, E. helioscopia, and G. chodoense.The intron of petB and rpl16 was absent in C. sabulosa, R. communis, and E. helioscopia.The rpl2 intron Fig.6CDS-based ML tree of Euphorbiaceae and Phyllanthaceae species was only absent in the plastome of C. sabulosa.We used Geneious Prime 2021.1.1 and the MAFFT alignment of cp.genomes from the nine species to compare the relative placements of genes across the species.The 192,904bp consensus sequence had 103,626 (54%) identical sites and 80% pair-wise identity.This analysis demonstrates that both family members have a close association, and C. sabulosa demonstrated a close link with both family members.These comparisons confirm the phylogenetic results and show that the chloroplast genome helps understand the evolutionary relationships within the Euphorbiaceae and Phyllanthaceae families.

Table 3
Described the comparison of C. sabulosa cp.genome with four Euphorbiaceae and four Phyllanthaceae species

Table 4
Genes showing elusive Ka/Ks ratio for eight selected species

Table 5
Ts, Tv substitutions, and Ts/Tv ratio (in IRs, SSC, and LSC) of selected Euphorbiaceae and Phyllanthaceae species

Table 6
The detailed analysis of InDels, Average InDel Length, InDel Diversity K(i), InDel Diversity per site Pi(i), and alignment length in LSC, IR, and SSC regions of C. sabulosa by making pairwise alignment with eight selected species of Euphorbiaceae and Phyllanthaceae families

Table 7
Six mutual highly polymorphic regions of selected Euphorbiaceae and Phyllanthaceae species